Dynamics and spatial organization of Kv1.3 at the immunological synapse of human CD4+ T cells.

Formation of the immunological synapse (IS) is a key event during initiation of an adaptive immune response to a specific antigen. During this process, a T cell and an antigen presenting cell form a stable contact that allows the T cell to integrate both internal and external stimuli in order to decide whether to activate. The threshold for T cell activation depends on the strength and frequency of the calcium (Ca2+) signaling induced by antigen recognition, and it must be tightly regulated to avoid undesired harm to healthy cells. Potassium (K+) channels are recruited to the IS to maintain the negative membrane potential required to sustain Ca2+ entry. However, the precise localization of K+ channels within the IS remains unknown. Here, we visualized the dynamic subsynaptic distribution of Kv1.3, the main voltage-gated potassium channel in human T cells. Upon T cell receptor engagement, Kv1.3 polarized toward the synaptic cleft and diffused throughout the F-actin rich distal compartment of the synaptic interface-an effect enhanced by CD2-CD58 corolla formation. As the synapse matured, Kv1.3 clusters were internalized at the center of the IS and released in extracellular vesicles. We propose a model in which specific distribution of Kv1.3 within the synapse indirectly regulates the channel function and that this process is limited through Kv1.3 internalization and release in extracellular vesicles.

S-acylation-dependent membrane microdomain localization of the regulatory Kvß2.1 subunit.

The voltage-dependent potassium (Kv) channel Kvß family was the first identified group of modulators of Kv channels. Kvß regulation of the α-subunits, in addition to their aldoketoreductase activity, has been under extensive study. However, scarce information about their specific α-subunit-independent biology is available. The expression of Kvßs is ubiquitous and, similar to Kv channels, is tightly regulated in leukocytes. Although Kvß subunits exhibit cytosolic distribution, spatial localization, in close contact with plasma membrane Kv channels, is crucial for a proper immune response. Therefore, Kvß2.1 is located near cell surface Kv1.3 channels within the immunological synapse during lymphocyte activation. The objective of this study was to analyze the structural elements that participate in the cellular distribution of Kvßs. It was demonstrated that Kvß peptides, in addition to the cytoplasmic pattern, targeted the cell surface in the absence of Kv channels. Furthermore, Kvß2.1, but not Kvß1.1, targeted lipid raft microdomains in an S-acylation-dependent manner, which was concomitant with peptide localization within the immunological synapse. A pair of C-terminal cysteines (C301/C311) was mostly responsible for the specific palmitoylation of Kvß2.1. Several insults altered Kvß2.1 membrane localization. Therefore, growth factor-dependent proliferation enhanced surface targeting, whereas PKC activation impaired lipid raft expression. However, PSD95 stabilized Kvß2.1 in these domains. This data shed light on the molecular mechanism by which Kvß2.1 clusters into immunological synapses during leukocyte activation.

Membrane Channels in Health and Diseases.

The goal of this Special Issue, entitled "Membrane Channels in Health and Diseases (https://www [...].

Abnormal innate and learned behavior induced by neuron-microglia miscommunication is related to CA3 reconfiguration.

Neuron-microglia communication through the Cx3cr1-Cx3cl1 axis is essential for the development and refinement of neural circuits, which determine their function into adulthood. In the present work we set out to extend the behavioral characterization of Cx3cr1-/- mice evaluating innate behaviors and spatial navigation, both dependent on hippocampal function. Our results show that Cx3cr1-deficient mice, which show some changes in microglial and synaptic terminals morphology and density, exhibit alterations in activities of daily living and in the rapid encoding of novel spatial information that, nonetheless, improves with training. A neural substrate for these cognitive deficiencies was found in the form of synaptic dysfunction in the CA3 region of the hippocampus, with a marked impact on the mossy fiber (MF) pathway. A network analysis of the CA3 microcircuit reveals the effect of these synaptic alterations on the functional connectivity among CA3 neurons with diminished strength and topological reorganization in Cx3cr1-deficient mice. Neonatal population activity of the CA3 region in Cx3cr1-deficient mice shows a marked reorganization around the giant depolarizing potentials, the first form of network-driven activity of the hippocampus, suggesting that alterations found in adult subjects arise early on in postnatal development, a critical period of microglia-dependent neural circuit refinement. Our results show that interruption of the Cx3cr1-Cx3cl1/neuron-microglia axis leads to changes in CA3 configuration that affect innate and learned behaviors.

Palmitoylation of Voltage-Gated Ion Channels.

Protein lipidation is one of the most common forms of posttranslational modification. This alteration couples different lipids, such as fatty acids, phospho- and glycolipids and sterols, to cellular proteins. Lipidation regulates different aspects of the protein's physiology, including structure, stability and affinity for cellular membranes and protein-protein interactions. In this scenario, palmitoylation is the addition of long saturated fatty acid chains to amino acid residues of the proteins. The enzymes responsible for this modification are acyltransferases and thioesterases, which control the protein's behavior by performing a series of acylation and deacylation cycles. These enzymes target a broad repertoire of substrates, including ion channels. Thus, protein palmitoylation exhibits a pleiotropic role by differential modulation of the trafficking, spatial organization and electrophysiological properties of ion channels. Considering voltage-gated ion channels (VGICs), dysregulation of lipidation of both the channels and the associated ancillary subunits correlates with the development of various diseases, such as cancer or mental disorders. Therefore, a major role for protein palmitoylation is currently emerging, affecting not only the dynamism and differential regulation of a moiety of cellular proteins but also linking to human health. Therefore, palmitoylation of VGIC, as well as related enzymes, constitutes a novel pharmacological tool for drug development to target related pathologies.

The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

The voltage-dependent potassium (Kv) channel Kv1.3 regulates leukocyte proliferation, activation, and apoptosis, and altered expression of this channel is linked to autoimmune diseases. Thus, the fine-tuning of Kv1.3 function is crucial for the immune system response. The Kv1.3 accessory protein, potassium voltage-gated channel subfamily E (KCNE) subunit 4, acts as a dominant negative regulatory subunit to both enhance inactivation and induce intracellular retention of Kv1.3. Mutations in KCNE4 also cause immune system dysfunction. Although the formation of Kv1.3-KCNE4 complexes has profound consequences for leukocyte physiology, the molecular determinants involved in the Kv1.3-KCNE4 association are unknown. We now show that KCNE4 associates with Kv1.3 via a tetraleucine motif situated within the carboxy-terminal domain of this accessory protein. This motif would function as an interaction platform, in which Kv1.3 and Ca2+/calmodulin compete for the KCNE4 interaction. Finally, we propose a structural model of the Kv1.3-KCNE4 complex. Our experimental data and the in silico structure suggest that the KCNE4 interaction hides a forward-trafficking motif within Kv1.3 in addition to adding a strong endoplasmic reticulum retention signature to the Kv1.3-KCNE4 complex. Thus, the oligomeric composition of the Kv1.3 channelosome fine-tunes the precise balance between anterograde and intracellular retention elements that control the cell surface expression of Kv1.3 and immune system physiology.-Solé, L., Roig, S. R., Sastre, D., Vallejo-Gracia, A., Serrano-Albarrás, A., Ferrer-Montiel, A., Fernández-Ballester, G., Tamkun, M. M., Felipe, A. The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

Remodeling of Kv7.1 and Kv7.5 Expression in Vascular Tumors.

Voltage-dependent potassium (Kv) channels contribute to the excitability of nerves and muscles. In addition, Kv participates in several cell functions, including cell cycle progression and proliferation. Kv channel remodeling has been associated with neoplastic cell growth and cancer. Kv7 channels are expressed in blood vessels, and they participate in the maintenance of vascular tone and are implicated in myocyte proliferation. Although evidence links Kv7 remodeling to different types of cancer, its expression in vascular tumors has never been studied. Endothelium-derived vascular neoplasms range from indolent lesions to highly aggressive and metastasizing cancers. Here, we show that Kv7.1 and Kv7.5 are evenly distributed in tunicas as well as the endothelium of healthy veins and arteries. The layered structure of vessels is lost in vascular tumors. By studying eight vascular tumors with different origins and characteristics, we found that Kv7.1 and Kv7.5 expression was changed in vascular cancers. While both channels were generally downregulated, Kv7.5 expression was clearly correlated with neoplastic malignancy. The vascular tumors did not contract; therefore, the role of Kv7 channels is probably related to proliferation rather than controlling vascular tone. Our results identify vascular Kv7 channels as targets for cancer detection and anticancer therapies.

Caveolar targeting links Kv1.3 with the insulin-dependent adipocyte physiology.

The voltage-dependent potassium channel Kv1.3 participates in peripheral insulin sensitivity. Genetic ablation of Kv1.3 triggers resistance to diet-induced weight gain, thereby pointing to this protein as a pharmacological target for obesity and associated type II diabetes. However, this role is under intense debate because Kv1.3 expression in adipose tissue raises controversy. We demonstrated that Kv1.3 is expressed in white adipose tissue from humans and rodents. Moreover, other channels, such as Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodel the Kv phenotype, which could lead to multiple heteromeric complexes, Kv1.3 markedly participates in the insulin-dependent regulation of glucose uptake in mature adipocytes. Adipocyte differentiation increased the expression of Kv1.3, which is targeted to caveolae by molecular interactions with caveolin 1. Using a caveolin 1-deficient 3T3-L1 adipocyte cell line, we demonstrated that the localization of Kv1.3 in caveolar raft structures is important for proper insulin signaling. Insulin-dependent phosphorylation of the channel occurs at the onset of insulin-mediated signaling. However, when Kv1.3 was spatially outside of these lipid microdomains, impaired phosphorylation was exhibited. Our data shed light on the putative role of Kv1.3 in weight gain and insulin-dependent responses contributing to knowledge about adipocyte physiology.

The Potassium Channel Odyssey: Mechanisms of Traffic and Membrane Arrangement.

Ion channels are transmembrane proteins that conduct specific ions across biological membranes. Ion channels are present at the onset of many cellular processes, and their malfunction triggers severe pathologies. Potassium channels (KChs) share a highly conserved signature that is necessary to conduct K⁺ through the pore region. To be functional, KChs require an exquisite regulation of their subcellular location and abundance. A wide repertoire of signatures facilitates the proper targeting of the channel, fine-tuning the balance that determines traffic and location. These signature motifs can be part of the secondary or tertiary structure of the protein and are spread throughout the entire sequence. Furthermore, the association of the pore-forming subunits with different ancillary proteins forms functional complexes. These partners can modulate traffic and activity by adding their own signatures as well as by exposing or masking the existing ones. Post-translational modifications (PTMs) add a further dimension to traffic regulation. Therefore, the fate of a KCh is not fully dependent on a gene sequence but on the balance of many other factors regulating traffic. In this review, we assemble recent evidence contributing to our understanding of the spatial expression of KChs in mammalian cells. We compile specific signatures, PTMs, and associations that govern the destination of a functional channel.

The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit.

The voltage-dependent K+ channel Kv1.3 (also known as KCNA3), which plays crucial roles in leukocytes, physically interacts with KCNE4. This interaction inhibits the K+ currents because the channel is retained within intracellular compartments. Thus, KCNE subunits are regulators of K+ channels in the immune system. Although the canonical interactions of KCNE subunits with Kv7 channels are under intensive investigation, the molecular determinants governing the important Kv1.3- KCNE4 association in the immune system are unknown. Our results suggest that the tertiary structure of the C-terminal domain of Kv1.3 is necessary and sufficient for such an interaction. However, this element is apparently not involved in modulating Kv1.3 gating. Furthermore, the KCNE4-dependent intracellular retention of the channel, which negatively affects the activity of Kv1.3, is mediated by two independent and additive mechanisms. First, KCNE4 masks the YMVIEE signature at the C-terminus of Kv1.3, which is crucial for the surface targeting of the channel. Second, we identify a potent endoplasmic reticulum retention motif in KCNE4 that further limits cell surface expression. Our results define specific molecular determinants that play crucial roles in the physiological function of Kv1.3 in leukocytes.

The Spanish Ion Channel Initiative (SICI) Consortium: Ten Years (2008⁻2018) of a Network of Excellence on Ion Channel Research.

The Spanish Ion Channel Initiative consortium (SICI, http://sici. [...].

D242N, a KV7.1 LQTS mutation uncovers a key residue for IKs voltage dependence.

KV7.1 and KCNE1 co-assemble to give rise to the IKs current, one of the most important repolarizing currents of the cardiac action potential. Its relevance is underscored by the identification of >500 mutations in KV7.1 and, at least, 36 in KCNE1, that cause Long QT Syndrome (LQTS). The aim of this study was to characterize the biophysical and cellular consequences of the D242N KV7.1 mutation associated with the LQTS. The mutation is located in the S4 transmembrane segment, within the voltage sensor of the KV7.1 channel, disrupting the conserved charge balance of this region. Perforated patch-clamp experiments show that, unexpectedly, the mutation did not disrupt the voltage-dependent activation but it removed the inactivation and slowed the activation kinetics of D242N KV7.1 channels. Biotinylation of cell-surface protein and co-immunoprecipitation experiments revealed that neither plasma membrane targeting nor co-assembly between KV7.1 and KCNE1 was altered by the mutation. However, the association of D242N KV7.1 with KCNE1 strongly shifted the voltage dependence of activation to more depolarized potentials (+50mV), hindering IKs current at physiologically relevant membrane potentials. Both functional and computational analysis suggest that the clinical phenotype of the LQTS patients carrying the D242N mutation is due to impaired action potential adaptation to exercise and, in particular, to increase in heart rate. Moreover, our data identify D242 aminoacidic position as a potential residue involved in the KCNE1-mediated regulation of the voltage dependence of activation of the KV7.1 channel.

An unconventional calmodulin-anchoring site within the AB module of Kv7.2 channels.

Calmodulin (CaM) binding to the AB module is crucial for multiple mechanisms governing the function of Kv7.2 (also known as KCNQ2) K(+) channel subunits, which mediate one of the main components of the non-inactivating K(+) M-current, a key controller of neuronal excitability. Structural analysis indicates that the CaM N-lobe engages with helix B, whereas the C-lobe anchors to the IQ site within helix A. Here, we report the identification of a new site between helices A and B that assists in CaM binding whose sequence is reminiscent of the TW helix within the CaM C-lobe anchoring site of SK2 K(+) channels (also known as KCNN2). Mutations that disrupt CaM binding within the TW site, helix B or helix A yield functional channels, whereas no function is observed when the TW site and helix A, or the TW site and helix B are mutated simultaneously. Our data indicate that the TW site is dispensable for function, contributes to the stabilization of the CaM-Kv7.2 complex and becomes essential when docking to either helix A or when helix B is perturbed.

Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

Involvement of potassium channels in the progression of cancer to a more malignant phenotype.

Potassium channels are a diverse group of pore-forming transmembrane proteins that selectively facilitate potassium flow through an electrochemical gradient. They participate in the control of the membrane potential and cell excitability in addition to different cell functions such as cell volume regulation, proliferation, cell migration, angiogenesis as well as apoptosis. Because these physiological processes are essential for the correct cell function, K+ channels have been associated with a growing number of diseases including cancer. In fact, different K+ channel families such as the voltage-gated K+ channels, the ether à-go-go K+ channels, the two pore domain K+ channels and the Ca2+-activated K+ channels have been associated to tumor biology. Potassium channels have a role in neoplastic cell-cycle progression and their expression has been found abnormal in many types of tumors and cancer cells. In addition, the expression and activity of specific K+ channels have shown a significant correlation with the tumor malignancy grade. The aim of this overview is to summarize published data on K+ channels that exhibit oncogenic properties and have been linked to a more malignant cancer phenotype. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

A non-canonical di-acidic signal at the C-terminus of Kv1.3 determines anterograde trafficking and surface expression.

Impairment of Kv1.3 expression at the cell membrane in leukocytes and sensory neuron contributes to the pathophysiology of autoimmune diseases and sensory syndromes. Molecular mechanisms underlying Kv1.3 channel trafficking to the plasma membrane remain elusive. We report a novel non-canonical di-acidic signal (E483/484) at the C-terminus of Kv1.3 essential for anterograde transport and surface expression. Notably, homologous motifs are conserved in neuronal Kv1 and Shaker channels. Biochemical analysis revealed interactions with the Sec24 subunit of the coat protein complex II. Disruption of this complex retains the channel at the endoplasmic reticulum. A molecular model of the Kv1.3-Sec24a complex suggests salt-bridges between the di-acidic E483/484 motif in Kv1.3 and the di-basic R750/752 sequence in Sec24. These findings identify a previously unrecognized motif of Kv channels essential for their expression on the cell surface. Our results contribute to our understanding of how Kv1 channels target to the cell membrane, and provide new therapeutic strategies for the treatment of pathological conditions.

Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology.

OBJECTIVE: Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. APPROACH AND RESULTS: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. CONCLUSIONS: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.

Kv1.3 in the spotlight for treating immune diseases.

INTRODUCTION: Kv1.3 is the main voltage-gated potassium channel of leukocytes from both the innate and adaptive immune systems. Channel function is required for common processes such as Ca2+ signaling but also for cell-specific events. In this context, alterations in Kv1.3 are associated with multiple immune disorders. Excessive channel activity correlates with numerous autoimmune diseases, while reduced currents result in increased cancer prevalence and immunodeficiencies. AREAS COVERED: This review offers a general view of the role of Kv1.3 in every type of leukocyte. Moreover, diseases stemming from dysregulations of the channel are detailed, as well as current advances in their therapeutic research. EXPERT OPINION: Kv1.3 arises as a potential immune target in a variety of diseases. Several lines of research focused on channel modulation have yielded positive results. However, among the great variety of specific channel blockers, only one has reached clinical trials. Future investigations should focus on developing simpler administration routes for channel inhibitors to facilitate their entrance into clinical trials. Prospective Kv1.3-based treatments will ensure powerful therapies while minimizing undesired side effects.

KCNE4-dependent modulation of Kv1.3 pharmacology.

The voltage-dependent potassium channel Kv1.3 is a promising therapeutic target for the treatment of autoimmune and chronic inflammatory disorders. Kv1.3 blockers are effective in treating multiple sclerosis (fampridine) and psoriasis (dalazatide). However, most Kv1.3 pharmacological antagonists are not specific enough, triggering potential side effects and limiting their therapeutic use. Functional Kv are oligomeric complexes in which the presence of ancillary subunits shapes their function and pharmacology. In leukocytes, Kv1.3 associates with KCNE4, which reduces the surface abundance and enhances the inactivation of the channel. This mechanism exerts profound consequences on Kv1.3-related physiological responses. Because KCNE peptides alter the pharmacology of Kv channels, we studied the effects of KCNE4 on Kv1.3 pharmacology to gain insights into pharmacological approaches. To that end, we used margatoxin, which binds the channel pore from the extracellular space, and Psora-4, which blocks the channel from the intracellular side. While KCNE4 apparently did not alter the affinity of either margatoxin or Psora-4, it slowed the inhibition kinetics of the latter in a stoichiometry-dependent manner. The results suggested changes in the Kv1.3 architecture in the presence of KCNE4. The data indicated that while the outer part of the channel mouth remains unaffected, KCNE4 disturbs the intracellular architecture of the complex. Various leukocyte types expressing different Kv1.3/KCNE4 configurations participate in the immune response. Our data provide evidence that the presence of these variable architectures, which affect both the structure of the complex and their pharmacology, should be considered when developing putative therapeutic approaches.